PROJECT SUMMARY It is clear that latently infected CD4+ T cells in HAART treated HIV-infected individuals are the major reservoir and obstacle to eradication. This research proposal specifically intends to address the FOA entitled Targeting Latently infected cells without reactivation with the following specific objective proposed by the FOA: to identify novel biomarkers of latently-infected cells that could be used to selectively target and kill cells harboring latent HIV-DNA. Current work has attempted to identify cell surface markers that preferentially harbor latent HIV, that is replication competent upon activation, however, these markers are not necessarily HIV-specific, at best can enrich for about 60% of the total HIV reservoir in CD4+ T cells, and often represent a substantial percentage of the total memory CD4+ T cell population, which would make targeted elimination of these cells as a therapy problematic. To circumvent this problem, we propose an unbiased approach where we will employ the non-conventional variable lymphocyte receptor (VLR) antibody platform of the evolutionary distant sea lamprey to isolate novel VLR antibodies that specifically target HIV-infected cells. We hypothesize that the large evolutionary distance of jawless vertebrates and the different protein architecture of VLR antibodies allows these proteins to detect antigens expressed by latently infected cells that conventional monoclonal antibodies cannot readily bind for structural or tolerogenic reasons. Optimism for success stems from our extensive experience in the generation of novel monoclonal VLR antibodies in lampreys that identify novel surface receptors of immune cell subsets that previously were not identified using standard methodologies. In Aim 1 we propose to use an in vitro model of HIV latency in primary CD4+ T cells to immunize lamprey larvae and then screen for monoclonal VLRs that can specifically bind to latently infected cells in vitro, using a high throughput assay system. In aim 2 we will tet a number of `candidate' VLRs that will be tested for their abilities to bind to and specifically deplete HIV-infected cells from ex vivo blood samples from HIV-infected individuals off and on HAART. In aim 2 we will test VLRs from a cohort of HIV-infected individuals who have been treated for variable duration to assess the generalizability of the VLRs ability to bind to latent cells. In aim 3, we will then characterize the protein or glycoprotein epitopes that are being recognized by VLRs using mass spectroscopy and biochemical methods. The current proposal has the potential to discover novel surface markers that can then be exploited to produce monoclonal agents that can more specifically target latently infected cells in vivo.